The practical utilization of regenerative medicine, which helps the regeneration of living tissues/organs that have fallen into functional disorder or functional incompetence, is currently proceeding. The regenerative medicine is novel medical technology of re-creating the same or similar forms or functions as in original tissues using 3 factors, i.e., cells, scaffolds, and growth factors, for living tissues that no longer recover by only natural healing ability possessed by organisms. In recent years, treatments using cells have been being gradually realized. Examples thereof include cultured epidermis using autologous cells, cartilage treatment using autologous cartilage cells, bone regeneration treatment using mesenchymal stem cells, cardiac muscle cell sheet treatment using myoblasts, corneal regeneration treatment using corneal epithelial sheets, and nerve regeneration treatment. These novel treatments, unlike conventional alternative medicine based on artificial materials (bone prosthetic materials or hyaluronic acid injection), help the repair or regeneration of living tissues and therefore produce high therapeutic effects. In fact, products such as cultured epidermis or cultured cartilage using autologous cells have been launched.
However, the problem of the variability of therapeutic effects or the problem of unexpected adverse effect has been in concern and has arisen actually in the treatments using cells as described above. Leading causes of such problems are considered to be the heterogeneity of cells obtained in culture and the nonuniform engraftment of cells at transplantation sites.
For example, it is known that cartilage cells tend to be dedifferentiated during the course of culture, and therefore, a culture method keeping them in a constant state has been demanded. Moreover, it is known that a cell generally changes its state depending on conditions under which the cell is present, particularly, cell density. For example, it is known that mesenchymal stem cells (MSCs), which are currently expected to be applied to treatments, tend to be differentiated into fat cells under low cell density conditions and, on the contrary, differentiated into osteocytes under high cell density conditions. For other cells as well, a very important factor is to control cell density at a constant level.
Meanwhile, many reports have demonstrated that a three-dimensional culture technique is important for efficiently culturing cells under conditions closer to an in vivo environment. However, it is known that when cells are inoculated to a three-dimensional matrix, the cells are not uniformly distributed in the matrix and are disproportionately located in the matrix. For the three-dimensional culture, it is therefore difficult to keep cell density constant. Although the three-dimensional cell culture presumably depends on the physical, biological and chemical properties of matrices, such as the adhesiveness of matrices to cells, the structure of matrices, and the hydrophilicity and hydrophobicity of matrices, it is generally difficult to uniformly distribute cells in a three-dimensional matrix and keep cell density constant. As a result, the state in which a cell state is not uniform in the three-dimensional matrix is formed, resulting in problems associated with cell homogeneity.
Moreover, for transplantation treatment using the obtained cells, the transplantation of cells cultured, for example, as in the atelocollagen gel-embedding culture of cartilage cells, together with a matrix is performed, in addition to the injection of a cell suspension. In this case, after culture, a construct consisting of a three-dimensional matrix and cells is trimmed appropriately for the shape of a transplantation site and transplanted thereto. If the cells are nonuniformly distributed in the three-dimensional matrix, nonuniform cell distribution also occurs in a fragment for transplantation obtained by trimming, generating a site strongly exhibiting therapeutic effects and a site hardly exhibiting therapeutic effects. Furthermore, the nonuniform cell density causes nonuniformity in the physical properties and physical strength of the transplanted fragment. Such nonuniformity becomes a factor responsible for the variability of therapeutic effects and reduction in the survival rate of the transplanted fragment and results in a lack of expected therapeutic effects. Furthermore, for aptitude required for the three-dimensional matrix as described above, it is important to be made of a material having biocompatibility, desirably biodegradability (because of the need for being spontaneously degraded in vivo), because of its use in transplantation. Unfortunately, there has not existed a three-dimensional matrix that satisfies all of these requirements.
As described above, from the viewpoint of regenerative medicine using cells, it is required that the three-dimensional matrix should be capable of uniformly distributing cells and retaining the cells in a state without nonuniformity both in a culture step and in a transplantation step and be also made of a biodegradable material because of the need for being applied to transplantation. Previous porous polymers, three-dimensional collagen matrices, or collagen gel-embedding culture have hardly overcome such a problem.
In general, living tissues are composed of cells and extracellular matrices (polymer constructs). Various life phenomena are consequences of their complicated interactions. Cells release various growth factors (drugs) and influence their own functions or the functions of other cells. On the other hand, the extracellular matrices secreted from the cells provide hydration space for cell functions, function as drug depots or scaffolds, and have significant influence on the functional manifestation or differentiation of cells.
The regenerative medicine, which has made remarkable progress in recent years, is gathering a lot of attention as highly advanced medicine that may substitute for artificial organs or organ transplantation. For realizing the regenerative medicine, it is important to wield each of principal factors in the regenerative medicine, i.e., cells, culture apparatuses, growth factors (drugs), and cell scaffolds (artificial extracellular matrices and materials).
Bone regeneration in the orthopedic or dental field is known as a region that is gathering a lot of attention in the regenerative medicine field. Bone diseases affecting legs or lower backs cause inability to walk, while bone diseases affecting teeth make dietary intake difficult. Thus, a bone disease causes remarkable reduction in QOL.
Infuse (combination of BMP-2 with a collagen sponge) treating spinal cord injury as well as BioOss (deproteinized bovine bone powder) and Gem21 (PDGF and βTCP) as bone prosthetic materials regenerating alveolar bone are known as current typical preparations for bone regeneration treatment. In general, [1] strength for structural maintenance, [2] securing of space for bone regeneration, [3] scaffolds for cells to regenerate bone, [4] induction of differentiation and growth of cells to regenerate bone, and [5] degradability associated with bone regeneration are known as properties required for preparations for bone regeneration treatment. Collagen, βTCP, or the like is widely used as a scaffold material for the preparations for bone regeneration treatment. Moreover, at the research level, bone regeneration has been performed by impregnating a gelatin (denatured form of collagen) sponge with bFGF or BMP-2 (Journal of Neurosurgery 91 851-856, 1999). Furthermore, studies have also been made on bone regeneration treatment using a therapeutic agent obtained by involving bone marrow mesenchymal stem cells in a scaffold matrix and culturing it (Motohiro Hirose and Hajime Ogushi, “Regenerative Medicine of Bone Using Regenerated Cultured Bone Tissues”, The Tissue Engineering 2007, 178-183; 2007.7).
The bone regeneration means that osteoblasts generate bone matrices to form hard bone consisting of osteocytes and the bone matrices, and is a phenomenon achieved by the osteoblasts. Not only the utilization of a cell graft (bone marrow mesenchymal stem cells, etc.) grown ex vivo but also the bone regenerating effect of growth factors (drugs) induces bone regeneration by the action on host-derived cells in a neighborhood thereof. For example, BMP differentiates undifferentiated mesenchymal cells into osteoblasts by its action and activates bone formation by the osteoblasts. Thus, since cells predominate in the bone regeneration, a scaffold material serving as a cell scaffold is very highly important.
Gelatin is well known as a typical scaffold material in general regenerative medicine. Gelatin is known as a material having high biocompatibility and high safety and has a good record with medical applications. Likewise, collagen is known as a material having a good record, but is lower soluble than gelatin and is largely limited by the concentration and pH of its solution (collagen cannot be prepared into a solution with a high concentration of dozens of %, a neutral solution, or the like). Hence, there are usually limitations on products into which collagen may be processed, prepared, or molded. Thus, a scaffold matrix using gelatin is desirable. However, there is a general perception that the gelatin matrix is less suitable as a scaffold material particularly for bone regeneration treatment. For example, it is disclosed that a gelatin sponge alone inhibits bone regeneration (Tabata, et al., Journal of Neurosurgery 91 851-856, 1999). Moreover, Ishii et al. (Dental Outlook, 97 (3), 665-677, 2001) have examined the influence of a collagen sponge and a gelatin sponge on the ability to regenerate alveolar bone and stated that gelatin lacks the ability to regenerate bone, compared with collagen.
In addition, since the bone regenerating power of a scaffold material alone is insufficient in case of collagen, ceramic materials, or synthetic polymers, their combinations with BMP, autologous blood, or autologous bone marrow mesenchymal stem cells have been reported under the present circumstances.
A cause of the absence of the favorable ability of these scaffold materials to regenerate bone has been considered to be insufficient “ability to infiltrate (introduce) cells into the scaffold matrix” or insufficient “ability to distribute or retain cells in the scaffold matrix”. The present inventors have thought that the absence of sufficient cell introducing power, distribution, or retaining power of the scaffold matrix as a cell support is responsible for not leading to bone regeneration.
For example, JP Patent Publication (Kokai) No. 62-122586 A (1987) discloses a porous support having a porosity of 40 to 95%, desirably 60 to 80% or lower, which consists of polyester or polypropylene. The porous support with the porosity cannot uniformly distribute or retain cells. Alternatively, JP Patent Publication (Kokai) No. 8-89239 A (1996) discloses a porous collagen sponge. It is known that the collagen sponge cannot uniformly distribute cells in a three-dimensional manner, possibly depending on its hydrophobicity or due to the heterogeneity of the material, though this cause is not clear. In addition, in the case where cells are cultured in advance and then transplanted, there also exists an approach called gel-embedding culture in which cells are embedded in a collagen gel and cultured. However, the cells are disproportionately located in the gel and nonuniformly distributed.